The overall goal of this research is to develop a mechanistic understanding of the vacuolar biogenesis pathways and endosome function in the yeast Saccharomycescerevisiae. Genetic analysis has led to the identification of a very large number of components required along the vacuolar assembly pathway in yeast, and two of these proteins, Vps55 and Vps68 are particularly of interest because these are integral membrane proteins that function at the endosome. Vps55 and Vps68 form a complex together with UipSp, a 25 kDa member of the large DUP240 gene family. The role of this integral membrane protein complex is being investigated as a docking/tethering site for the large collection of soluble Vps proteins that are recruited to the Golgi and endosome membrane during protein sorting and vacuole membrane traffic. "Class E" proteins proteins function at the endosome in the formation of the multi-vesicular body (MVB) and the sorting of cargo proteins into these intralumenal vesicles. Ten of these Vps proteins form three separate ESCRT protein complexes (Endosome Sorting Complex Required for Transport), and the AAA- ATPase Vps4 is required to disassemble the ESCRT complexes. We are investigating the role of three other Class E Vps proteins that are required to recruit the Vps4 complex to the endosome and to stimulate ESCRT disassembly by activating the Vps4 ATPase activity. We will also exploit our new genetic screen for mutants defective for membrane invagination at the endosome;analysis of these genes should reveal important insight into the mysterious process of MVB membrane invagination. We are investigating the connection between endosome function and the ability of yeast to sense glucose in the extracellular environment. Some of the yeast Class E mutants are Sucrose NonFermenting (Snf-), and only a subset of each of the ESCRT complex polypeptides are required for yeast to be Snf+. The high-affinity glucose sensor in yeast, Snf3, localizes to MVBs in yeast mutants defective for MVB function. Studies of membrane traffic in yeast have resulted in a deeper understanding of membrane transport in all eukaryotic cells, and these studies in yeast are also providing important insights into our understanding of lysosomal storage diseases and the cellular requirements for HIV viral particle formation.